Gas phase sample preparation for cryo-electron microscopy

ABSTRACT

The present invention provides methods for controllably forming a layer of amorphous ice and other amorphous solids on a substrate, and also provides cryo-electron microscopy (cryo-EM) sample preparation methods and systems that utilize in vacuo formation of amorphous ice and other solids. Formation of the amorphous solid layer can be independent of the deposition of sample molecules to be analyzed using electron microscopy, and allows for the generation of a uniformly thick layer. Optionally, mass spectrometry instruments are used to generate and purify molecules deposited on the generated amorphous solid layer. The techniques and systems described herein can deliver near ideal cryo-EM sample preparation to greatly increase resolution, sensitivity, scope, and throughput of cryo-EM protein imaging, and therefore greatly impact the field of structural biology.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/626,250, filed Dec. 23, 2019, which is a U.S. National StageApplication filed under 35 U.S.C. § 371 of International Application No.PCT/US2018/041120, filed Jul. 6, 2018, which claims the benefit of U.S.Provisional Patent Application No. 62/529,778, filed Jul. 7, 2017. Theseapplications are hereby incorporated by reference in their entireties.

This invention was made with government support under GM118110 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

X-ray crystallography has traditionally been the standard for providingstructural analysis for biological research, and while X-raycrystallography still provides high resolution structural information,this technique has several downsides. One such limitation is that alarge amount of sample is required for X-ray crystallography. This makesX-ray crystallography impractical in cases where it is difficult togenerate large, highly pure quantities of the target molecule.

Single particle cryo-electron microscopy (cryo-EM) is emerging as apowerful alternative for structural studies of eukaryotic cells,proteins (>150 kDa), and macromolecular complexes (e.g., liposomes,organelles, and viruses) (Stark et al., Microscopy, 2016, 65(1):23-34)).Cryo-EM is unique, providing 3D structural information onnon-crystalline specimens while also often requiring smaller sampleamounts than X-ray crystallography. With the development of a new classof electron detector and advances in software image reconstruction,cryo-EM has approached atomic level resolution, enabling many newbiological discoveries. This technology has driven considerableinterest, but it still has a number of limitations, including resolutionthat is still a factor of four less than what is theoretically possible(Glaeser, R. M., Nature Methods, 2016, 13(1):28-32). The majority oflimitations are due to sample preparation, which typically requirespurification and vitrification. Encasing the sample in vitreous ice(i.e., amorphous ice) helps protect the sample from radiation damagefrom the electron microscope. Ideal sample vitrification would involveuse of an amorphous ice layer, just thick enough to accommodate theparticle(s) of interest.

However, in practice, the process of sample vitrification is far fromideal. Typical sample preparation techniques involve solubilization ofprotein analytes in water followed by pipetting onto a hydrophilic EMgrid. The grid is blotted with filter paper (removing >99.99% of thesample) and then plunged into a bath of cryogen, vitrifying theremaining water/sample. A key component to obtaining proper 3D data fromEM structures is that the particles must all be of the same structuralconformation but randomly oriented within the amorphous ice. Proper 3Danalysis is completed via reconstruction of a number of images, thusrequiring numerous particles randomly oriented in the same structuralconformation. Unfortunately, currently existing sample preparationtechniques impart a preferred orientation of the particles (due largelyto particle migration to the air/water interface), and the particlesoften become deformed/stretched at the air-water interface, therebydestroying the required structural heterogeneity. Similar deformationsarise from the absorption of the sample to the EM grid substrate. Formany cases, the imaged structural heterogeneity does not exist innature, and without a means of separating the multiple conformations,cryo-EM often cannot be utilized to obtain 3D information (Glaeser, R.M., Nature Methods, 2016, 13(1):28-32; and Yu et al., J. Structuralbiology, 2014, 187:1-9).

Another requirement of an ideally vitrified specimen is to have a highdensity of randomly oriented identical molecules located within a holeof the cryo-EM grid. This imposes a significant problem as conventionalpreparation techniques yield only a very few particles per grid hole.Several factors contribute to this issue. First is that the majority ofthe sample gets removed during the blotting process. Highly concentratedsamples can help compensate for this problem. However, even at thehighest concentrations very few particles are observed per grid hole, asthey preferentially absorb to the EM-grid, leaving the holes lessoccupied. Second, formation of ice in conventional methods causes theice at the center of a hole in the grid to be thinner than the ice nearthe edges, forcing the particles to the outer edges. This process canalso impart a preferred orientation, especially if the molecules arethicker in one dimension than another. With only a few analyte particlesper hole, data acquisition times must be extended and data files becomevery large (i.e., greater than 5 Tb), since much of the EM grid must beimaged to generate sufficient signal for analysis. Beyond limiting thescope and types of proteins that can be analyzed, conventionalapproaches puts a significant strain on the computational resourcesrequired to analyze the data Cheng et al., Cell, 2015, 161(3):438-449).

For these reasons, it is desirable to obtain improved cryo-EM samplepreparation methods and systems so as to improve the applicability andaccuracy of cryo-EM.

SUMMARY OF THE INVENTION

The present invention provides new methods for controllably forming alayer of amorphous ice and other frozen amorphous solids on a substrate,and also provides cryo-EM sample preparation methods and systems thatutilize in vacuo vitrification. In certain aspects of the invention,formation of an amorphous solid layer is independent of the depositionof a sample to be analyzed, thus allowing for the generation ofuniformly thick layer. Furthermore, the formation of an amorphous solidlayer may be monitored during formation and any imperfection in thesolid layer may be corrected using ion milling or related techniques. Incertain aspects, the present invention is also able to provide cryo-EMsample preparation resulting in increased image resolution, decreasedimage acquisition time, and increased sensitivity.

Certain aspects of the invention further include the use of massspectrometry to purify analyte particles, including but not limited toproteins, protein complexes, and cells, in the gas-phase for subsequentvitrification. Samples prepared in this way can be extracted from themass spectrometer using a cryo-transfer sample holder and placeddirectly into an EM for imaging. One implementation of this methodutilizes a modified mass spectrometer that allows for gas-phasepurification of analyte ions. The ions will be passed over a cooledsample probe where they are deposited onto an EM sample holder andvitrified.

Amorphous solids, or non-crystalline solids, refer to solids that lackthe long-range molecular order characteristic of crystals. For example,ice formed using the methods and systems described herein is preferablyvitreous ice (also referred to herein as amorphous ice). Common H₂O iceis a hexagonal crystalline material where the molecules are regularlyarranged in a hexagonal lattice. In contrast, vitreous ice lacks theregularly ordered molecular arrangement. Vitreous ice and the otheramorphous solids available with the present invention are producedeither by rapid cooling of the liquid phase (so the molecules do nothave enough time to form a crystal lattice) or by compressing ordinaryice (or ordinary solid forms) at very low temperatures.

One embodiment of the present invention provides a method for preparinga sample for cryo-electron microscopy (cryo-EM) comprising the steps offorming a vapor stream of atoms or molecules, and directing the vaporstream toward a substrate surface such that the atoms or moleculesimpinge on the substrate surface while under vacuum. The substratesurface is at a temperature of −100° C. or less (optionally at atemperature of −150° C. or less, −175° C. or less, or −195° C. or less).As a result, a layer of an amorphous solid is formed on the surface ofthe substrate. The method further comprises forming an analyte beamcontaining charged or uncharged analyte particles to be analyzed usingEM; and contacting the amorphous solid layer with the analyte beam. Theanalyte particles are embedded on or within the deposited amorphoussolid layer thereby forming a suitable sample for EM analysis.

As used in embodiments described herein, “under a vacuum” refers to apressure of 10⁻⁴ Torr or less, a pressure 10⁻⁵ Torr or less, or apressure 10⁻⁶ Torr or less. In embodiments, the atoms or molecules ofthe vapor stream contact the substrate surface, and the step ofcontacting the amorphous solid layer with the analyte beam, are carriedout at a pressure equal to or less than 10⁻⁴ Torr, 10⁻⁵ Torr, or 10⁻⁶Torr.

The analyte particles forming the analyte beam can be charged oruncharged particles depending on the deposition method used to depositthe molecules onto the ice layer. Preferably, the analyte particles andthe molecules making the amorphous solid layer are substantiallyrandomly orientated when deposited on the substrate, such as on amembrane, film, or EM grid. The analyte beam can be an ion beam,molecular beam, or particle beam. In an embodiment, the analyteparticles are ions formed using techniques including, but not limitedto, electrospray ionization and laser desorption, such asmatrix-assisted laser desorption/ionization (MALDI). Preferably, theanalyte particles are ionized under native electrospray conditions so asnot to perturb structural conformation of the particles. In a furtherembodiment, the analyte ions are formed using a mass spectrometer whichoptionally isolates or purifies the analyte ions. Alternatively, theparticle beam is a molecular beam. In a further embodiment, themolecular beam is produced by creating an aerosol of an analyte particlecontaining solution and introducing the aerosol into the vacuum system.

In certain embodiments, the analyte beam is characterized by anintensity selected from the range of 0.025 to 25 particles per 1 μm² persecond, 0.05 to 10 particles per 1 μm² per second, or 0.1 to 5 particlesper 1 μm² per second. In certain embodiments, the analyte beam ischaracterized by a spot size selected from the range of 800 μm² to 3.8E7μm².

The analyte particles can be purified or isolated, such as by a massspectrometer device, before being deposited onto the amorphous solid.Preferably, the analyte beam is characterized by a purity of at least85%, 90%, 95%, or 99%. For analyte particles, such as proteins, whichmay have significant conformational structures, it is desirable that theanalyte beam is characterized by a conformation purity of at least 85%,90%, 95% or 99%. For example, it may be desirable to analyze thestructure of a particular protein as expressed in a cell. Accordingly,it is necessary to provide an EM sample where all or most of the proteinanalyte molecules retain the same conformational structure.

Analyte particles useful with the present invention include, but are notlimited to, protein molecules, multi-protein complexes, protein/nucleicacid complexes, nucleic acid molecules, virus particles,micro-organisms, sub-cellular components (e.g., mitochondria, nucleus,Golgi, etc.), and whole cells. In some embodiments, the analyteparticles are molecular entities, single molecules, or multiplemolecules complexed together through non-covalent interactions (such ashydrogen bonds or ionic bonds). In embodiments, the analyte particleshave a molecular mass exceeding 1,000 Daltons, 10,000 Daltons, 50,000Daltons, 100,000 Daltons, or 150,000 Daltons.

The deposited analyte particles may be embedded within the amorphoussolid layer, deposited on the surface, or both. Since it is preferablefor EM to have the analyte particles fully encased within the amorphoussolid, a further embodiment comprises additionally contacting theamorphous solid layer with the atoms or molecules from the vapor streamafter the analyte particles have been deposited on the amorphous solidlayer. This will provide an additional layer of the solid on top of thedeposited molecules. Alternatively, a further method comprisescontacting the amorphous solid layer with the analyte beam concurrentlywith contacting the substrate surface with the atoms or molecules fromthe vapor stream. In a further embodiment, the vapor stream (ormolecular water beam) is reflected off of one or more reflectingsurfaces prior to contacting the substrate surfaced. This ensures thatthe atoms or molecules are broken up and have a randomized orientationbefore contacting the substrate.

In an embodiment, the vapor stream is generated using a Knudsen-typeeffusion cell, a molecular beam doser, or a co-effusion of a matrix withanalyte into the system. Optionally, the vapor stream is characterizedby an intensity selected from the range of 4.8E9 to 2.8E11 molecules perμm² per second and/or a spot size selected from the range of 800 μm² to3.8E7 μm². In an embodiment, the vapor stream comprises a flux ofmolecules having a uniformity within 98% over an area of 7 mm². Incertain embodiments, the vapor stream is a molecular beam. Molecularbeams are streams of molecules traveling in the same or similardirection and can be produced by allowing a gas at higher pressure toexpand through a small orifice into a chamber at lower pressure to formthe beam. Preferably, the incident trajectory of the particles of thevapor stream contacting the substrate surface is within 1 degree ofnormal to the substrate surface.

Preferably the vapor stream is controlled, or the deposited amorphoussolid layer is milled, etched, or otherwise refined, so that theamorphous solid layer has a thickness of 2 microns or less, 150 nm orless, or 100 nm or less. Preferably, the amorphous solid layer has auniform thickness which does not vary by more than 5% across thesubstrate. Preferably, the layer of the amorphous solid has an extent ofcrystallinity less than or equal to 1%. Preferably, the layer of theamorphous solid has a purity of at least 85%, 90%, 95%, or 99%.

The vapor stream can comprise any molecules or atoms able to formamorphous solids where exposed to extremely low temperatures andpressures. Such molecules and atoms include, but are not limited to,cyclohexanol, methanol, ethanol, isopentane, water, O₂, Si, SiO₂, S, C,Ge, Fe, Co, Bi and mixtures thereof. Optionally, the vapor streamcomprises charged molecules.

In an embodiment, the vapor stream comprises water molecules. Thus, anembodiment of the present invention provides a method for preparing asample for cryo-electron microscopy (cryo-EM) comprising the steps offorming a molecular beam of water molecules, and contacting a surface ofa substrate with the molecular water beam while under vacuum. Thesubstrate surface is at a temperature of −100° C. or less (optionally ata temperature of −150° C. or less, −175° C. or less, or −195° C. orless). As a result, a layer of amorphous ice is formed on the surface ofthe substrate. The method further comprises forming an analyte beamcontaining charged or uncharged analyte particles to be analyzed usingEM; and contacting the deposited ice layer with the analyte beam. Themolecular beam is controlled, or the deposited ice is milled, etched, orotherwise refined, so that the deposited ice layer with the analyteparticles has a thickness of 2 microns or less, preferably 150 nm orless, or 100 nm or less. Preferably, the ice layer has a uniformthickness which does not vary by more than 5% across the substrate.

In an embodiment, the analyte particles are ions and the analyte sourceis able to generate a controllable ion beam containing charged analyteions (such as electrospray ion deposition) and direct the ion beam tocontact the receiving surface of the cryo-EM probe. In a furtherembodiment, the system further comprises a modified mass spectrometerthat can provide purified ions to the analyte source. In anotherembodiment, the system comprises an electron microscope where thecryo-EM probe is directly transferred from the deposition portion of theinstrument to the microscope portion of the instrument for analysis.

In certain embodiments, the formation of the vitreous ice layer or otheramorphous solid layer is monitored (alone or in conjunction with thedeposition of the analyte particles) to ensure that the proper thicknessis achieved and that the layer deposited on the substrate is amorphousas opposed to crystalline. For example, a microscale is utilized toconfirm that a solid (such as ice) is being uniformly deposited on thesubstrate, continuous film, continuous membrane, cryo-EM grid or probe.Similarly, the vacuum chamber contains a window or other means to allowoptical light or infrared light to illuminate the sample. An optical orinfrared light detection cell able to receive light transmitted orreflected from the deposited solid layer is then used to determine ifthe solid is amorphous or crystalline. Preferably, the monitoring of theformation of the amorphous solid is performed in real time.

In an embodiment, the present invention provides a cryo-electronmicroscopy (cryo-EM) sample preparation system comprising: a) a vacuumchamber; b) a cryo-EM probe positioned with the vacuum chamber, whereinthe cryo-EM probe comprises a receiving surface; c) a beam doser able toproduce a controllable molecular beam (or vapor stream) and direct themolecular beam (or vapor stream) to contact the receiving surface of thecryo-EM probe; d) a temperature control means able to provide atemperature of −100° C. or less to the receiving surface of the cryo-EMprobe; and e) an analyte source in fluid communication with the vacuumchamber, wherein the analyte source is able to produce a controllableanalyte beam containing charged or uncharged analyte particles anddirect the analyte beam to contact the receiving surface of the cryo-EMprobe. In an embodiment, the molecular beam comprises cyclohexanol,methanol, ethanol, isopentane, water, O₂, Si, SiO₂, S, C, Ge, Fe, Co,Bi, or mixtures thereof. In an embodiment, the beam doser is able toproduce a controllable molecular water beam. Optionally, the vacuumchamber is able to provide a pressure of 10⁻⁴ Torr, a pressure of 10⁻⁵Torr, or a pressure of 10⁻⁶ Torr. Optionally, the temperature controlmeans is able to provide a temperature of −150° C. or less, −175° C. orless, or −195° C. or less. Devices and methods for providing a vacuumchamber or other kind of surface at cryogenic temperatures are wellknown in the art. For example, in an embodiment, the temperature controlmeans comprises a cold finger able to provide localized temperaturecontrol of the receiving surface of the cryo-EM probe.

Optionally, the substrate described in the embodiments provided hereinis an electron microscopy (EM) grid as known in the art. The EM grid maycomprise a metal, including but not limited to copper, rhodium, nickel,molybdenum, titanium, stainless steel, aluminum, gold, or combinationsthereof as known in the art. Additionally, the EM grid may comprise acontinuous film or membrane which is positioned across the top or bottomsurface of the grid, or within the holes of the grid, so as to provide asolid support for the formation of the amorphous solid. Preferably, theEM grid is covered by a thin film or membrane which includes, but is notlimited to, films and membranes comprising graphene, graphene oxide,silicon oxide, silicon nitride, carbon, and combinations thereof. With agrid that does not contain a film or membrane, the molecular beamintended to form the amorphous solid may pass through at least a portionof the holes in the grid without producing a suitable layer. The film ormembrane should be thin enough so as to not scatter electrons.Preferably, the film or membrane has an approximate thickness or 15 nmor less, 10 nm or less, 5 nm or less, 2 nm or less, or 1 nm or less. Inan embodiment, the substrate is an EM grid comprising a graphene orgraphene oxide monolayer film or membrane positioned across the surfaceof the grid.

An embodiment of the present invention provides a method for forming alayer of vitreous ice on a substrate comprising the steps of: a) forminga molecular beam of water molecules; b) while under vacuum (i.e., at apressure of 10⁻⁴ Torr or less, a pressure 10⁻⁵ Torr, or less or apressure 10⁻⁶ Torr or less), contacting the substrate with the molecularwater beam, wherein the substrate is at a temperature of −100° C. orless (optionally at a temperature of −150° C. or less, −175° C. or less,or −195° C. or less), thereby forming a layer of vitreous ice on thesubstrate. Preferably the deposited ice layer has a thickness of 2microns or less, 150 nm or less, or 100 nm or less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cryo-electron microscopy (cryo-EM) sample preparationsystem in an embodiment of the present invention.

FIG. 2 shows a partial cross section of the system of FIG. 1 and furtherdepicts the path taken the analyte beam and molecular water beam.

FIG. 3 shows an ion analyte source used in an embodiment of theinvention, where the analyte source comprises ions optics (such as andskimmers) to focus and direct an ion beam.

FIG. 4 , panel a), shows a beam doser able to produce a controllablemolecular water beam in an embodiment of the invention. Panel b) shows across section of a beam doser.

FIG. 5 shows a cross-section of an instrument including a beam doser anda sample holder, as well as an infrared light beam and microscale usedto monitor the amorphous ice formed on the probe.

FIG. 6 shows a transmission electron microscopy (TEM) image of amorphousice formed on a graphene oxide support film supported by a copper/goldgrid. The graphene oxide support film has a thickness less than 1 nm.Holes in the support grid having amorphous ice can be clearlydistinguished from holes or regions in the grid without any formation ofamorphous ice.

FIG. 7 shows an additional TEM image of amorphous ice formed on agraphene oxide support film, where a portion of the ice layer is foldedback over itself.

FIG. 8 shows an additional TEM image of amorphous ice formed on agraphene oxide support film. This image also shows a hole drilledthrough the amorphous ice with an electron beam, as well as regionshaving no ice and crystalline ice.

FIG. 9 shows a TEM image of crystalline ice formed on the grid with ahole drilled through the crystalline ice using an electron beam.

FIG. 10 shows a TEM image of amorphous ice formation on a support gridwithout the use of a support film covering the grid.

FIG. 11 , top graph, shows IR spectra of amorphous H₂O ice deposited atT<70K (dashed line) and T>70 (solid line). The bottom graph showsspectra of crystalline H₂O ice deposited at 20K (solid line), 80K(dot-dashed line), and 150K (dotted line). These spectra were obtainedfrom Mastrapa et al., Icarus, 2008, 197:307-320.

FIG. 12 shows IR spectra of crystalline hexagonal ice (bottom line) andamorphous ice (top line) obtained using the present invention.

FIG. 13 illustrates the rate of growth of the amorphous ice by measuringfrequency. The physical characteristics of the quartz crystal usedresulted in a 21 Hz decrease in resonance frequency for every 1 nmthickness of ice which forms on its surface. From 20 minutes to 50minutes, the rate of ice formation in this experiment was 1.94 nm/min.

FIG. 14 shows a schematic of a cryo-electron microscopy (cryo-EM) samplepreparation system similar to the embodiment shown in FIG. 1 . Thisembodiment comprises a secondary pumping system for making the vaporstream and a storage tank able to directly store the sample after theamorphous solid has been formed on the surface of the sample.

FIG. 15 illustrates a cryo probe within the vacuum chamber positioned toreceive the analyte beam and molecular beam from the doser.

FIG. 16 illustrates a cross-section of a cryo probe within a vacuumchamber as shown in FIG. 15 .

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the present invention provides novel cryo-EMsample preparation methods that utilize in vacuo vitrification able to:(1) increase image resolution, (2) decrease image acquisition time, and(3) allow for many orders of magnitude increase in sensitivity.Optionally, readily available mass spectrometry instruments can be usedto purify proteins and protein complexes in the gas-phase for subsequentin vacuo vitrification. Samples prepared in this way can be extractedfrom the mass spectrometer—using a cryo-transfer sample holder andplaced directly into an EM for imaging.

Example 1—Cryo-EM Sample Preparation Instruments

FIGS. 1-5 and 14-16 show exemplary cryo-electron microscopy (cryo-EM)sample preparation systems 25 according to certain embodiments of thepresent invention. A cryo-EM probe 2 able to hold or contain a sample isinserted into vacuum chamber 1. The temperature of the system ismaintained using a coolant, such as liquid nitrogen, which is stored intank 8 and transferred through cold finger 5, while one or more turbopumps 9 are used to maintain the vacuum.

Analyte particles are collected in an analyte source 6 where they arefocused into an analyte beam 13 (such as through electrospray iondeposition) and directed to contact the sample plate being held bycryo-EM probe 2 (see FIG. 2 ). FIG. 3 shows one type of an analytesource 6 where analyte ions or molecules are drawn into the analytesource 6 through capillary 16. One or more ion optic devices, such asskimmers 17, are used to focus the analyte ions into a beam 13 and tocontrol the release speed of the ions through exit aperture 18.

A beam doser 4 is used to generate one or more 14 molecular beams (suchas molecular water beams) and direct the molecular beam down to thecryo-EM probe 2. Vapor 34 (such as water vapor) used to generate themolecular beams 14 is transported through a heated transfer line 19 andthen transported along the same axis as travelled by the analyte beam13. Optionally, the molecular beams 14 are reflected off a series ofreflecting surfaces 26, which breaks up the molecules and randomizestheir orientation (see FIG. 4 , panel b). Microchannel plate 20 onlyallows reflected molecular beams 14 to pass through if the beams areorientated along the proper direction (i.e., in a direction co-axialwith the analyte beam 13). It should be noted that while these examplesspecify water vapor and

The cryo-EM probe 2 is used to laterally move the sample as needed. Forexample, the sample holder 21 can move the probe 2 to a quartz crystalmicroscale 10 which is used to monitor the build-up of the ice layer onthe cryo-EM probe 2 (see FIGS. 1 and 5 ). Additionally, the sample canbe moved to an infrared (IR) sample plate 15 which is illuminated by anIR light beam 22 provided by a fiber optic IR light source 7. Thetransmitted light is collected by the optical detection cell 11 andtransmitted to the fiber optic IR spectrometer 23 to monitor whether thedeposited ice layer comprises vitreous ice or crystalline ice.

In another example, FIGS. 14-16 illustrate a cryo-electron microscopy(cryo-EM) sample preparation system 25 where the system comprises an asecondary pumping system 27 used for making the vapor stream 34, and astorage tank 28 able to directly store the sample from the vacuumchamber 1 after the vitreous ice has been formed on the sample. Thestorage tank is kept low temperatures and can also utilize coolants suchas liquid nitrogen.

Example 2—Gas-Phase Analyte Purification

Protein ions can be purified in the gas-phase, collected in vacuo, and,once removed from the vacuum, retain their enzymatic function (Blake etal., Analytical Chemistry, 2004, 76(21):6293-6305). Following theseexperiments, a mass spectrometer was modified so that analyte proteinions could be purified and deposited directly onto a sample probe. Theprobe surface originally comprised glycerol on stainless steel; however,with the requested cryo-transfer probe, the purified protein ions aredeposited directly onto a cryogenic EM grid that has previously beencovered in vitreous ice. Formation of the ice layer independent of thesample allows for the generation uniformly thick ice. Any imperfectionin the ice may be corrected using ion milling or related techniques.Independently forming the ice also allows for appropriate qualitycontrol measures prior to committing the sample.

Vitreous ice undergoes a phase transition from high density amorphous(HDA) ice at atmospheric pressure where it is formed, to low densityamorphous (LDA) ice at the low pressures of the mass spectrometer(Mishima et al., Nature, 1998, 396(6709): 329-335). It is essential thatthe purified protein ions have structures that are reflective of thatfrom solution. Ion mobility experiments have shown that under nativespray conditions this is achieved (Seo et al., AngewandteChemie-International Edition, 2016, 55(45): 14173-14176). Additionally,ion/ion chemical reactions are also used to reduce the charge of apurified protein population prior to deposition, and thereby restoresolution phase structure.

Example 3—Formation of Amorphous Ice

The present invention provides methods and instruments for preparingsamples of an analyte with amorphous solids for use in cryo-electronmicroscopy (cryo-EM). The amorphous solids, which protect the analytefrom radiation damage and dehydration during imaging, must remaintransparent to the electron beam during EM. This requires the amorphoussolid layer (e.g., the ice layer) be thin, on the order of the samethickness of the molecules to be analyzed, and the solid must beamorphous. If the amorphous solid becomes too thick, the electrons maybe scattered causing defocusing and reduction in image contrast. Ifordered crystals, such as crystalline ice, begin to form, the electronswill be diffracted and the resulting diffraction pattern will obscurethe image (Cheng et al., Cell, 2015, 161(3): 438-449). The difficultiesassociated with forming vitrified sample as described above are wellknown in the art.

Probably less well known is the importance that vitreous ice plays inthe outer solar system and interstellar space (Fama et al., SurfaceScience, 2008, 602(1): 156-161; and Cleeves et al., Science, 2014,345(6204): 1590-1593). The high vacuum and coldness of space provides anatural forming ground for vitreous ice. In fact, it is the most commonform of ice outside our solar system (Guillot et al., J. ChemicalPhysics, 2004, 120(9):4366-4382). The obvious difficulties of studyingvitreous ice have required the development of techniques to formamorphous ice in the laboratory under interstellar conditions, inparticular, in cryogenic vacuum chambers. Many of the simulatedconditions require the formation of very thin amorphous ice layers. Thishas been accomplished through the use of Knudsen-type effusion cells andmolecular beam dosers placed within the vacuum system (Moeller et al.,Optical Engineering, 2012, 51(11): 115601; and Huffstetler et al.,Journal of Vacuum Science & Technology a-Vacuum Surfaces and Films,2001, 19(3):1030-1031). However, these methods do not describe how toprepare amorphous solids in conjunction with a sample to be analyzed.

The formation of amorphous solids in conjunction with mass spectrometryinstruments and techniques as described herein provide a novel means ofpreparing vitreous samples for cryo-EM which removes all the limitationsassociated with the currently employed plunge freezing method. In oneembodiment, an uncovered cryo-EM grid or a cryo-EM grid covered by athin continuous film or membrane held at liquid nitrogen temperatures isused as a landing surface within a mass spectrometer. Examples of EMgrids with continuous films or membranes across the surface of the gridcan be obtained from Quantifoil Micro Tools GmbH (Groβlöbichau, Germany)and Electron Microscopy Sciences (Hatfield, Pa., USA). The grid (coveredor uncovered) is populated with biomolecules utilizing gas-phase analytepurification techniques described above. Within the same vacuum chamberis a molecular beam doser aimed at the landing surface. The job of thedoser is to produce a controllable molecular beam of water which impactsthe cryo-surface/grid, forming vitreous ice (for general descriptions ofdosers, see Guillot et al., J. Chemical Physics, 2004, 120(9):4366-4382;Moeller et al., Optical Engineering, 2012, 51(11): 115601; and Westly etal., J. Chemical Physics, 1998, 108(8):3321-3326).

Initially a very thin layer of ice is generated on the substrate. Thisis followed by deposition of analyte particles, either directly from ananalyte source or after gas-phase purification using a massspectrometer. Concomitantly with the collection of the analyteparticles, the molecular water beam is used to encase the analyteparticles in amorphous ice. Alternately, the analyte particles arelanded on an initial amorphous ice surface and then covered/encased withamorphous ice using the vapor stream. Buildup of ice is monitored inreal time using quartz crystal microbalances (Moeller et al., OpticalEngineering, 2012, 51(11): 115601; and Gutzler et al., Review ofScientific Instruments, 2010, 81(1): 015108). When collection/samplepreparation is complete, the probe and substrate are removed from thedevice and transferred directly into a cryo-EM.

FIGS. 6-9 show transmission electron microscopy (TEM) images ofamorphous ice formed on a graphene oxide support film supported bycopper and/or gold grids. The vitreous ice was collected at atemperature of −175° C., with a 15 minute exposure to the molecular beamdoser under vacuum. The resulting ice layer was approximately 15 micronsthick. Hexagonal ice was obtained the same manner, with the exceptionthat the temperature was −155° C. As seen in the accompanying figures,holes 40 in the support grid covered by regions of amorphous ice 41 canbe clearly distinguished from regions or holes in the grid without anyformation of ice 42. Holes 47 were also drilled through the amorphousice regions 41 crystalline ice regions 43 with an electron beam.

As seen in FIGS. 8 and 9 , amorphous ice regions 41 appear verydifferent from the regions of crystalline hexagonal ice 43. As a whole,these figures demonstrate that a layer of a solid was formed over theholes in the grid, where the layer was not the typical crystalline formof the solid.

These observations were further confirmed through infrared (IR)spectrometry. FIG. 11 shows IR spectra of amorphous H₂O ice andcrystalline H₂O ice deposited on a substrate (Mastrapa et al., Icarus,2008, 197:307-320). As can be seen in these spectra, the peaks from theamorphous ice shifted to shorter wavelengths compared to the peaks fromthe crystalline ice. FIG. 12 shows IR spectra of crystalline hexagonalice (bottom line) and amorphous ice (top line) using the presentinvention. The peaks of the amorphous ice obtained under the presentinvention shifted to shorter wavelengths as compared to the crystallineice, similar to what was reported in Mastrapa et al. Accordingly, it isbelieved these results clearly indicate that amorphous ice wassuccessfully deposited on the sample.

Additionally, FIG. 13 illustrates the rate of growth of the amorphousice by measuring the frequency of a quartz crystal microbalance. Thephysical characteristics of the quartz crystal microbalance resulted ina 21 Hz decrease in resonance frequency for every 1 nm thickness of icewhich forms on its surface. Accordingly, from 20 minutes to 50 minutes,the rate of ice formation in this experiment was 1.94 nm/min

While the above experiments utilized a thin continuous graphene oxidelayer over a copper or gold support grid, successful formation ofamorphous ice was also observed in grids which did not utilize amembrane or film over the grid. For example, FIG. 10 shows a grid 44without the use of a continuous film or membrane. Water molecules couldsimply pass through at least a portion of the holes in the grid. As aresult, there may be little or no ice bridging the empty holes 46.However, a layer of ice spanning several holes 45 was still seen.

Example 4—Formation of Other Amorphous Solids

The vitreous ice, which protects analytes from radiation damage anddehydration during imaging, must remain transparent to the electron beamduring EM. This requires the ice layer be thin, on the order of the samethickness of the molecules to be analyzed, and the ice must beamorphous. If the amorphous ice becomes too thick, the electrons may bescattered causing defocusing and reduction in image contrast. Ifcrystalline ice begins to form, the electrons will be diffracted and theresulting diffraction pattern will obscure the image (Cheng et al.,Cell, 2015, 161(3): 438-449).

In addition to water, there are other substances which will formamorphous solids at cold temperatures. These include, but are notlimited to cyclohexanol, methanol, ethanol, isopentane, O₂, SiO₂, S, C,Ge, Fe, Co, and Bi, among many others. As with water, the amorphousstate is obtained through condensation from the gas phase. Unlike water,which can be transformed to an amorphous solid by several techniques,these elements require vapor-condensation to form in the non-crystallinestate (Zallen R., The Physics of Amorphous Solids, 1983, 8-10). Briefly,a vapor stream of the matrix in question is formed by heating (thermalvaporization), vaporization by electron beam, vaporization by ionbombardment, or by plasma-induced decomposition, all in vacuum. Thevacuum chamber contains a cold surface onto which the atoms condense.Their thermal energy is extracted before they can migrate to thecrystalline conformation. The result is a thin film (<50 micron thick)of amorphous solid.

The fact that substances such as (but not limited to) Si, Ge, Fe, Co,and Bi require vapor condensation to form, the incorporation of thesecompounds with the instrument of Example 1 provides a novel means ofpreparing vitreous samples for cryo-EM with matrices other than ice. Inaddition to the matrix materials used, the porosity/density of theamorphous material being formed can be controlled through the depositionangle employed as well as the energy of the deposited matrix molecules(Dohnalek et al., Journal of Chemical Physics, 2003, 118(1): 364-372).This novel capability will enable fine tuning of the amorphous materialto provide maximum protection to the biomolecules during cryo-EManalysis. In one instance, a cryo-EM grid (either uncovered or a gridcovered by a thin film or membrane) held at liquid nitrogen temperaturesis used as a landing surface within the sample preparation instrument(Example 1). The grid is populated with biomolecules utilizingelectrospray deposition. Within the same vacuum chamber is avaporization source aimed at the landing surface. This source may beplaced off axes to effect the angle of incidence of the matrix moleculeson the surface. The job of the vaporization source is to produce acontrollable vapor stream of material (including but not limited to H₂O,Si, Ge, Fe, Co, Bi) that impacts the cryo-surface/grid, forming andamorphous solid.

Initially a very thin layer of material is generated on the grid. Thisis followed by collection of macromolecules isolated by the ESIdeposition source. Concomitantly with the collection of these molecules,the vapor stream is used to encase the sample. When collection/samplepreparation is complete, the probe and EM grid are removed from thesample preparation instrument and transferred directly into the cryo-EM.

Having now fully described the present invention in some detail by wayof illustration and examples for purposes of clarity of understanding,it will be obvious to one of ordinary skill in the art that the same canbe performed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

When a group of materials, compositions, components or compounds isdisclosed herein, it is understood that all individual members of thosegroups and all subgroups thereof are disclosed separately. Everyformulation or combination of components described or exemplified hereincan be used to practice the invention, unless otherwise stated. Whenevera range is given in the specification, for example, a temperature range,a time range, or a composition range, all intermediate ranges andsubranges, as well as all individual values included in the ranges givenare intended to be included in the disclosure. Additionally, the endpoints in a given range are to be included within the range. In thedisclosure and the claims, “and/or” means additionally or alternatively.Moreover, any use of a term in the singular also encompasses pluralforms.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements.

One of ordinary skill in the art will appreciate that startingmaterials, device elements, analytical methods, mixtures andcombinations of components other than those specifically exemplified canbe employed in the practice of the invention without resort to undueexperimentation. All art-known functional equivalents, of any suchmaterials and methods are intended to be included in this invention. Theterms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.Headings are used herein for convenience only.

All publications referred to herein are incorporated herein to theextent not inconsistent herewith. Some references provided herein areincorporated by reference to provide details of additional uses of theinvention. All patents and publications mentioned in the specificationare indicative of the levels of skill of those skilled in the art towhich the invention pertains. References cited herein are incorporatedby reference herein in their entirety to indicate the state of the artas of their filing date and it is intended that this information can beemployed herein, if needed, to exclude specific embodiments that are inthe prior art.

The invention claimed is:
 1. A method for preparing a sample forcryo-electron microscopy (cryo-EM) comprising the steps of: a) forming avapor stream of atoms or molecules and directing the vapor stream towarda substrate surface under vacuum and at a cryogenic temperature, therebyforming an amorphous solid layer of the atoms or molecules; and b)forming an analyte beam containing charged or uncharged analyteparticles; and directing the analyte beam toward the substrate surface,thereby embedding the analyte particles on or within the amorphous solidlayer deposited on the substrate surface.
 2. The method of claim 1,wherein the analyte beam and vapor stream are directed toward thesubstrate surface concurrently.
 3. The method of claim 1 comprisingdepositing atoms or molecules from the vapor stream after the analyteparticles have been deposited on the substrate surface.
 4. The method ofclaim 1, wherein the analyte beam is directed toward the substratesurface after the amorphous solid layer has been formed.
 5. The methodof claim 1 comprising purifying the analyte particles and generating theanalyte beam using a mass spectrometer.
 6. The method of claim 1,wherein the analyte beam is an ion beam is generated using electrosprayionization or laser desorption.
 7. The method of claim 1, wherein theanalyte beam is a molecular beam produced by creating an aerosol of ananalyte particle.
 8. The method of claim 1, wherein the analyte beam isa particle beam.
 9. The method of claim 1, wherein the vapor streamcomprises water molecules.
 10. The method of claim 1, wherein the vaporstream is comprised of charged molecules.
 11. The method of claim 1,wherein the vapor stream comprises molecules or atoms able to formamorphous solids, said molecules or atoms comprising one or more ofcyclohexanol, methanol, ethanol, isopentane, water, O₂, Si, SiO₂, S, C,Ge, Fe, Co, and Bi.
 12. The method of claim 1, wherein the vapor stream,analyte beam, or both are directed to the substrate surface at apressure equal to or less than 10⁻⁴ Torr.
 13. The method of claim 1,wherein the vapor stream, analyte beam, or both are directed to thesubstrate surface at a pressure equal to or less than 10⁻⁵ Torr.
 14. Themethod of claim 1 further comprising the step of analyzing said analyteparticles on or within the amorphous solid layer using a cryo-electronmicroscope.
 15. The method of claim 1, wherein the analyte particles aremolecular entities, single molecules, or multiple molecules complexedtogether through non-covalent interactions.
 16. The method of claim 1,wherein the analyte particles are protein molecules, multi-proteincomplexes, protein/nucleic acid complexes, nucleic acid molecules, virusparticles, micro-organisms, sub-cellular components, or whole cells. 17.The method of claim 1, wherein the amorphous layer has a thickness of 2microns or less.
 18. The method of claim 1, wherein the vapor stream isgenerated using a Knudsen type effusion cell, a molecular beam doser ora co-effusion of a matrix with analyte into the system.
 19. The methodof claim 1, wherein the substrate surface is at a temperature of −100°C. or less.
 20. The method of claim 1, wherein the layer of theamorphous solid has an extent of crystallinity less than or equal to 1%.